The responses to light, odorants, chemoattractants, and over one hundred hormones and neurotransmitters are mediated by G-protein coupled receptors. These receptors activate heterotrimeric G-proteins which in turn stimulate or inhibit adenylyl cyclases, phosphodiesterases, phospholipases, and ion channels. Desensitization is associated with phosphorylation of the receptors by specific kinases. Current views hold that the seven transmembrane helices of a G-protein coupled receptor form a compact bundle spanning the membrane and that agonist binding alters critical interactions between the helices, transmitting a conformational change to the cytoplasmic loops. The family of cAMP chemoattractant receptors (cARs) in Dictyostelium provides an excellent model system for investigating the mechanisms by which agonists induce the transition to an activated state and the pathways involved in desensitization. A combined genetic and biochemical approach is planned to investigate the structure and function of the cARs. Methods are described to screen randomly mutagenized receptors and identify residues required for high affinity binding and appropriate agonist specificity. To delineate the mechanism of activation, a variety of strategies are presented to isolate high affinity receptors that fail to elicit responses as well as constitutively active and hypersensitive receptors. Substitutions which cause agonist-independent phosphorylation, shift the activation curve to the left of the binding curve, or increase the basal activity of the receptor are anticipated. Wild-type and mutant cARs will be purified and reconstituted for structural analysis in two-dimensional crystals. Investigations of the conventional phosphorylation dependent as well as a distinct phosphorylation independent mechanism of desensitization are planned. Analysis of serine cluster substitutions and disruption of the cAR kinase gene will elucidate the role of receptor phosphorylation in desensitization of adenylyl cyclase activation and chemotaxis. Reconstitution studies of signal transduction mutants are intended to identify which downstream component is modified to attenuate adenylyl cyclase activity. Original screens of cell lines carrying random genomic insertions, generated by restriction enzyme mediated integration (REMI), are expected to uncover new genes in each of these desensitization pathways.